Adipose tissue is a type of connective tissue found in many places in the body - around internal organs, bone marrow, breast tissue, and under the skin. Adipose tissue stem cells (ASCs) are obtained easier than other types of multipotent mesenchymal stem cells (MSCs). That is why subcutaneous tissue can be harvested during various procedures, including minimally invasive procedures such as liposuction. During liposuction, 100 ml to 3000 ml of adipose tissue is obtained, which contains about 1x106 mesenchymal stem cells/200 ml of fat[1]. The process of cell isolation, the target of the study, provides the basis for further use of SVF (Stromal Vascular Fraction), which can differentiate into other cell types, including adipocytes, chondroblasts, myoblasts and osteoblasts[2].
The fat collected from the patient is placed in sterile bottles and transported to the laboratory in a thermos containing cooling cartridges. The isolation process should begin by pouring the material into 500ml conical centrifuge tubes and centrifuging at an acceleration of 1200-2000g for 5 minutes. After the first centrifugation, two fractions appear in the conical centrifuge tube - the upper fraction is fat, and the lower fraction contains, among other things, blood. Above the upper fraction is a layer of oil, which should be removed. The upper fraction (fat) should be transferred to a new tube, and then Collagenase II solution (1g in 10ml PBS) is added to the homogenized tissue in a ratio of 1:1 or 1:2, later centrifuge at an acceleration of 1260g for 5 minutes. Incubation of fat in the Collagenase II solution is done horizontally, on a rocker, and should last about 40-60 minutes at 37°C. During the incubation, the connections between cells are destroyed. After this time, the tube should be centrifuged again at an acceleration of 1360g for 5 minutes. The next step is to pellet the top layer of tissue to access the bottom layer. On its bottom, after rinsing with DMEM (Dulbecco's Modified Eagle Medium) and centrifuging 400g for 5 minutes, a pellet containing SVF that can initiate multipotent stromal cell culture or be used as a finished therapeutic product. After pelleting the supernatant, the resulting pellet should be resuspended in the complete mesenchymal stem cell culture media and then transferred to a culture bottle. The culture should be incubated at 37°C under 5% CO2, which will allow the cells to multiply correctly and adhere to the plastic bottom of the bottle.
The above-described process is the result of the development of a universal scheme for the isolation of stem cells for their further use in cell therapies. Examples of the use of these cells include the treatment of hard-to-heal wounds, extensive burns, osteoarthritis, or nerve injuries[3].